A wealth of epidemiological evidence indicates that ovarian hormones play a crucial role in the etiology of breast cancer (Kelsey et al., Epidemiol. Rev. 15:36-47 (1993)). Specifically, the observations that early menarche, late menopause and postmenopausal hormone replacement therapy are each associated with increased breast cancer risk, whereas early oophorectomy is associated with decreased breast cancer risk, have led to the hypothesis that breast cancer risk is proportional to cumulative estradiol and progesterone exposure (Henderson et al, Cancer Res. 48:246-253 (1988); Pike et al., Epidemiol. Rev. 15:17-35 (1993)). As such, elucidating the mechanisms by which hormones contribute to mammary carcinogenesis is a central goal of breast cancer research.
In addition to their roles in the pathogenesis of breast cancer, estradiol and progesterone are the principal steroid hormones responsible for regulating the development of the mammary gland during puberty, pregnancy and lactation (Topper et al., Physiol. Rev. 60:1049-1106 (1980)). For example, estradiol action is required for epithelial proliferation and ductal morphogenesis during puberty, whereas progesterone action is required for ductal arborization and alveolar differentiation during pregnancy (Bocchinfuso et al., J. Mamm. Gland Biol. Neoplasia, 2:323-334 (1997); Humphreys et al., J. Mamm. Gland Biol. Neoplasia, 2:343-354 (1997); Topper et al., 1980). The effects of estradiol and progesterone in a given tissue are ultimately determined by the activation and repression of their respective target genes.
Protein kinases represent the largest class of genes known to regulate differentiation, development, and carcinogenesis in eukaryotes. Many protein kinases function as intermediates in signal transduction pathways that control complex processes such as differentiation, development, and carcinogenesis (Birchmeier et al., BioEssays, 15:185-190 (1993); Bolen, Oncogene, 8:2025-2031 (1993); Rawlings et al., Immunol. Rev., 138:105-119 (1994)). Accordingly, studies of protein kinases in a wide range of biological systems have led to a more comprehensive understanding of the regulation of cell growth and differentiation (Bolen, 1993; Fantl et al., Annu. Rev. Biochem., 62:453-481(1993); Hardie, Symp. Soc. Exp. Biol., 44:241-255 (1990)).
Not surprisingly, aberrant expression or mutations in several members of the protein kinase family have been reportedly involved in the pathogenesis of cancer both in humans and in rodent model systems (Cardiff et al., Cancer Surv., 16:97-113 (1993); Cooper, Oncogenes, Publ., Jones & Bartlett, Boston, Mass., (1990); DiFiore et al., Cell, 51:1063-1070 (1987); Muller et al, Cell, 54:105-115 (1988)). Protein kinases function as molecular switches in signal transduction pathways that regulate cellular processes, such as proliferation and differentiation. In addition, some protein kinases are expressed in a lineage-specific manner, and are therefore useful markers for defining cellular subtypes (Dymecki et al., Science, 247:332-336 (1990); Mischak et al., J. Immunol., 147:3981-3987 (1991); Rawlings et al, 1994; Schnurch et al., Development. 19:957-968 (1993); Siliciano et al., Proc. Natl. Acad. Sci. USA, 89:11194-11198 (1992); Valenzuela et al., Neuron, 15:573-584 (1995)).
A key role played by serine/threonine kinases in regulating diverse cellular processes is exemplified by studies of SNF1-related kinases. The SNF1 family of protein kinases is composed of at least two subfamilies. The first subfamily includes SNF1 and its plant homologues including NPK5, AKin10, BKIN12, and Rkin1, as well as the mammalian SNF1 functional homologue, AMPK (Alderson et al., Proc. Natl. Acad. Sci. USA, 88:8602-8605 (1991); Carling et al., J. Biol. Chem., 269 11442-11448 (1994); LeGuen et al., Gene, 120:249-254 (1992); Muranaka et al., Mol. Cell. Biol., 14:2958-2965 (1994)). More recently, additional mammalian SNF1-related kinases have been identified that define a second subfamily. These include C-TAK1/p78 (involved in cell cycle control), MARK1, MARK2/Emk, SNRK (involved in adipocyte differentiation), and Msk (involved in murine cardiac development), as well as the C. elegans kinase, PAR-1 (Becker et al., Eur. J. Biochem., 235:736-743 (1996); Drewes et al., Cell, 89:297-308 (1997); Peng et al., Science, 277:1501-1505 (1997); Peng et al., Cell Growth Differ., 9:197-208 (1998); Ruiz et al., Mech. Dev., 48:153-164 (1994)). Less closely related to either subfamily are Wpk4, Melk, and KIN1, SNF1-related kinases found in wheat, mice, and Schizosaccharomyces pombe, respectively (Heyer et al., Mol. Reprod. Dev., 47:148-156 (1997); Levin et al., Proc. Natl. Acad. Sci. USA, 87:8272-8276 (1990); Sano et al., Proc. Natl. Acad. Sci. USA, 91:2582-2586 (1994)).
SNF1 is composed of a heterotrimeric complex that is activated by glucose starvation and is required for the expression of genes in response to nutritional stress (Carlson et al., Genetics, 98:25-40 (1981); Celenza et al., Mol. Cell. Biol., 9:5045-5054 (1989); Ciriacy, Mol. Gen. Genet., 154:213-220 (1977); Fields et al., Nature, 340:245-246 (1989); Wilson et al., Curr. Biol., 6:1426-1434 (1996); Yang et al., Science, 257:680-682 (1992); Yang et al., EMBO J., 13:5878-5886 (1994); Zimmermann et al., Mol. Gen. Genet., 154:95-103 (1977); Hardie et al., Semin. Cell Biol., 5: 409-416 (1994)). In fact, SNF-1 itself has been found to mediate cell cycle arrest in response to starvation (Thompson-Jaeger et al., Genetics, 129:697-706 (1991)).
Like SNF1, the mammalian SNF1-related kinase, AMPK, is involved in the cellular response to environmental stresses, particularly those that elevate cellular AMP:ATP ratios. Once activated, AMPK functions to decrease energy-requiring anabolic pathways, such as sterol and fatty acid synthesis while up-regulating energy-producing catabolic pathways such as fatty acid oxidation (Moore et al., Eur. J. Biochem., 199:691-697 (1991); Ponticos et al., EMBO J., 17:1688-1699 (1998)). AMPK complements the snf1 mutation in yeast and phosphorylates some of the same targets as SNF1 (Hardie, Biochem. Soc. Symp., 64:13-27 (1999); Hardie et al., Biochem. Soc. Trans., 25:1229-1231 (1997); Hardie et al., Biochem. J., 338:717-722 (1999); Woods et al., J. Biol. Chem., 271:10282-10290 (1996)). Like SNF1, AMPK is a heterotrimer composed of a, b, and g subunits that are homologous to the subunits of SNF1 (Hardie, 1999). Thus, AMPK and SNF1 are closely related both functionally and structurally, demonstrating that the regulatory pathways in which they operate have been highly conserved during evolution.
For instance, C-TAK1/p78 appears to be involved in cell cycle regulation based on its ability to phosphorylate and inactivate Cdc25c (Peng et al., 1997; Peng et al., 1998). Since Cdc25c controls entry into mitosis by activating cdc2, inactivation of Cdc25c by C-TAK1 would be predicted to regulate proliferation negatively. Consistent with this model, C-TAK1/p78 is down-regulated in adenocarcinomas of the pancreas (Parsa, Cancer Res. 48: 2265-2272 (1988)).
Perhaps the most compelling evidence that SNF1 kinases are involved in development is the observation that mutations in the C. elegans SNF1-related kinase, PAR-1, result in an inability to establish polarity in the developing embryo (Guo et al., Cell, 81:611-620 (1995)). Specifically, par-1 mutations disrupt P granule localization, asymmetric cell divisions, blastomere fates, and mitotic spindle orientation during early embryogenesis.
In an analogous manner, the mammalian PAR-1 homologue, MARK2/Emk, is asymmetrically localized in epithelial cells in vertebrates, and expression of a dominant negative form of MARK2 disrupts both cell polarity and epithelial cell—cell contacts (Bohm et al., Curr. Biol., 7:603-606 (1997)). In addition, overexpression of either MARK2 or its close family member MARK1 results in hyperphosphorylation of microtubule-associated proteins, disruption of the microtubule array, and cell death (Drewes et al 997). Thus, members of the SNF1 kinase family have been demonstrated to regulate a variety of important cellular processes.
In light of these findings, it is clear that prior to the present invention, there was a need to identify and study the role of protein kinases in mammary development and carcinogenesis, as well as provide insight into the regulation of pregnancy-induced changes in the mammary tissue that occur in response to estrogen and progesterone.